Mechanical Loading and Bone

描述（由申请人提供）：本研究的长期目标是阐明负荷诱导骨重塑的机制，并开发独特的以负荷为基础的治疗方法来预防骨质流失。基于我们最近的观察，本研究的具体目标是确定膝关节的机械负荷（膝关节负荷-对膝关节施加轻微的侧向负荷）如何不仅在负载（现场）骨中而且在非负载（远程）骨中发挥破骨细胞发育的整体抑制作用。作为潜在的调控机制，我们将重点关注分泌因子（如Wnt3a、NGF′、TNF′等）和低密度脂蛋白受体相关蛋白5 （Lrp5）介导的信号传导。在母项目中，我们已经证明膝关节负荷通过髓内压力的振荡调节增强胫骨和股骨的骨形成。然而，其对骨吸收的影响尚不清楚。利用小鼠去卵巢模型模拟绝经后骨质疏松症的初步研究表明，膝关节负荷可以抑制骨髓细胞的多核破骨细胞的发育，而且这种负荷效应不仅在负荷股骨中观察到，而且在未负荷的对侧股骨中也观察到。在这个竞争性更新项目中，我们将验证关节负荷（膝盖/肘部负荷）可以通过lrp5介导的Wnt信号传导（Wnt3a作为分泌因子）以及与其他分泌因子的相互作用，以系统的方式抑制ovx诱导的破骨细胞发生的假设。为了验证这一假设，我们提出了两个特定的目标，使用小鼠负荷模型（膝关节负荷、肘部负荷、尺骨弯曲和胫骨负荷），并测定骨重塑和原代骨髓细胞。目的1：确定关节负荷对破骨细胞发生的局部和整体影响目的2：评估负荷调节分泌因子在破骨细胞发生中的作用为了响应机械负荷，我们将进行x射线成像和集落形成单位测定。我们还将检测血清中关键分泌因子如Wnt3a、NGF、TNF、OPG、RANKL等的表达。将培养原代骨髓细胞，并研究负荷驱动的破骨细胞发生调控机制。我们将检查调节因子的表达，包括NFATc1（破骨细胞生成的主转录因子）和破骨细胞标志物，如OSCAR，组织蛋白酶K等。我们将使用Lrp5 KO小鼠（全局的，有条件地选择骨细胞），以及中和抗体、RNA干扰（功能丧失）和质粒（功能获得）。我们期望这个项目将有助于我们对骨吸收的负荷驱动调节的基本理解，并有助于全球预防骨质流失的负荷方案的发展。

DESCRIPTION (provided by applicant): The long-term objective of this study is to elucidate the mechanisms underlying loading-induced bone remodeling and develop unique loading-based therapies for preventing bone loss. The specific goal of this study, based on our most recent observations, is to determine how mechanical loading to the knee (knee loading - application of mild lateral loads to the knee) may exert global suppression of osteoclast development not only in the loaded (on-site) bone but also in the non-loaded (remote) bone. As a potential regulatory mechanism, we will focus on secretory factors (e.g., Wnt3a, NGF�, TNF�, etc.) and low-density lipoprotein receptor-related protein 5 (Lrp5) mediated signaling. In the parent project, we have shown that knee loading enhances bone formation in the tibia and the femur through the oscillatory modulation of intramedullary pressure. However, its effects on bone resorption have not been well understood. Preliminary studies using a mouse ovariectomized model, which mimics post-menopausal osteoporosis, indicate that knee loading can suppress development of multi-nucleated osteoclasts from bone marrow cells, and the loading effects are observed not only in the loaded femur but also in the non-loaded contralateral femur. In this competitive renewal project, we will test the hypothesis that joint loading (knee/elbow loading) can suppress an OVX-induced osteoclastogenesis in a systemic manner through Lrp5-mediated Wnt signaling with Wnt3a as a secretory factor, as well as interactions with other secretory factors. To examine this hypothesis, we propose two specific aims using a mouse loading model (knee loading, elbow loading, ulna bending, and tibia loading), and assays for bone remodeling and primary bone marrow cells. Aim 1: Determine the local and global effects of joint loading on osteoclastogenesis Aim 2: Evaluate the role of load-modulated secretory factors in osteoclastogenesis In response to mechanical loading, we will conduct X-ray imaging and colony forming unit assays. We will also examine expression of critical secretory factors such as Wnt3a, NGF�, TNF�, OPG, RANKL, etc. in the serum. Primary bone marrow cells will be cultured, and the mechanisms underlying loading-driven regulation of osteoclastogenesis will be investigated. We will examine expression of regulatory factors, including NFATc1 (master transcription factor for osteoclastogenesis) and osteoclast markers such as OSCAR, cathepsin K, etc. We will employ Lrp5 KO mice (global, and conditionally selective to osteocytes), as well as neutralizing antibodies and RNA interference (loss of a function), and plasmids (gain of a function). We expect that this project will contribute to our basic understanding of load-driven regulation of bone resorption and development of loading regimens useful for global prevention of bone loss.